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A combination of Let-7d, Let-7g and Let-7i serves as a stable reference for normalization of serum microRNAs.

Chen X, Liang H, Guan D, Wang C, Hu X, Cui L, Chen S, Zhang C, Zhang J, Zen K, Zhang CY - PLoS ONE (2013)

Bottom Line: Recent studies have indicated that circulating microRNAs (miRNAs) in serum and plasma are stable and can serve as biomarkers of many human diseases.A combination of let-7d, let-7g and let-7i is selected as a reference for the normalization of serum miRNAs and it is statistically superior to the commonly used reference genes U6, RNU44, RNU48 and miR-16.This has important implications for proper experimental design and accurate data interpretation.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Engineering Research Center for microRNA Biology and Biotechnology, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu, China.

ABSTRACT
Recent studies have indicated that circulating microRNAs (miRNAs) in serum and plasma are stable and can serve as biomarkers of many human diseases. Measurement of circulating miRNAs with sufficient sensitivity and precision, however, faces some special challenges, among which proper normalization is the most critical but often an underappreciated issue. The primary aim of this study was to identify endogenous reference genes that maintain consistent levels under various conditions to serve as an internal control for quantification of serum miRNAs. We developed a strategy combining Illumina's sequencing by synthesis (SBS) technology, reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay, literature screening and statistical analysis to screen and validate the most suitable reference genes. A combination of let-7d, let-7g and let-7i is selected as a reference for the normalization of serum miRNAs and it is statistically superior to the commonly used reference genes U6, RNU44, RNU48 and miR-16. This has important implications for proper experimental design and accurate data interpretation.

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Related in: MedlinePlus

Characterization of the absolute concentration and the stability of let-7d/g/i in serum.(A) Dynamic range and sensitivity of the RT-qPCR assay for measuring let-7d/g/i (n = 5). Synthetic single-stranded let-7d/g/i ranging from 0.01 attomole (0.0033 attomole each, equivalent to 6×103 copies in total) to 10 pmol (3.3 pmol each) were serially diluted over ten orders of magnitude and were assessed by the RT-qPCR assay. The resulting Cq values were plotted versus the amount of input let-7d/g/i to generate a standard curve. An assay using water instead of RNA for reverse-transcription was included as a negative control. (B) Correlation of serum volume to the Cq values (n = 5). Total RNA was extracted from different volumes of serum ranging from 10 µL to 400 µL. The levels of serum let-7d/g/i were assessed by RT-qPCR. The resulting Cq values were plotted versus the serum volume used for RNA extraction. An assay using water instead of RNA for reverse-transcription was included as a negative control. (C) Stability of let-7d/g/i in serum after extended storage (n = 5). Serum samples were equally divided and stored at room temperature, 4°C, -20°C or -80°C for 1, 2, 3, 7, 14 or 30 days. For each time point, total RNA was isolated and let-7d/g/i was measured by RT-qPCR assay. Storage at room temperature for 30 days yielded no apparent increase in Cq values. (D) Instability of other RNAs in serum (n = 5). Serum samples were equally divided and stored at room temperature for 1 to 24 h. For each time point, total RNA was isolated, and the levels of some large molecular weight RNA (β-actin, GAPDH and 28S rRNA) and snRNA/snoRNA (U6, RNU44, RNU48, SNORD24, SNORD38B, SNORD43, SNORA66 and SNORA74A) were measured by RT-qPCR. Storage at room temperature for 24 h resulted in an apparent increase of Cq values for these RNAs. (E) Stability of let-7d/g/i in serum after RNase digestion (n = 5). Serum samples were treated with 10 U/ml RNase A and 400 U/ml RNase T1 for 4 h at 37°C. After the treatment, the RNA was extracted from the serum, and the levels of let-7d/g/i were assessed by RT-qPCR. (F) Instability of other RNAs in serum after RNase digestion (n = 5). Serum samples were treated with 10 U/ml RNase A and 400 U/ml RNase T1 for 1, 2 or 4 h at 37°C. After the treatment, the RNA was extracted and the levels of the indicated RNAs were assessed by RT-qPCR assay. (G and H) Stability of let-7d/g/i under acidic or alkaline conditions (n = 5). Serum samples were incubated for 1 h under acidic (pH 2) or alkaline (pH 12) conditions. The levels of let-7d/g/i were assessed by RT-qPCR. (I) Stability of let-7d/g/i in serum following re-freezing and re-thawing of the samples (n = 6). Serum samples were subjected to eight freeze-thaw cycles.
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pone-0079652-g004: Characterization of the absolute concentration and the stability of let-7d/g/i in serum.(A) Dynamic range and sensitivity of the RT-qPCR assay for measuring let-7d/g/i (n = 5). Synthetic single-stranded let-7d/g/i ranging from 0.01 attomole (0.0033 attomole each, equivalent to 6×103 copies in total) to 10 pmol (3.3 pmol each) were serially diluted over ten orders of magnitude and were assessed by the RT-qPCR assay. The resulting Cq values were plotted versus the amount of input let-7d/g/i to generate a standard curve. An assay using water instead of RNA for reverse-transcription was included as a negative control. (B) Correlation of serum volume to the Cq values (n = 5). Total RNA was extracted from different volumes of serum ranging from 10 µL to 400 µL. The levels of serum let-7d/g/i were assessed by RT-qPCR. The resulting Cq values were plotted versus the serum volume used for RNA extraction. An assay using water instead of RNA for reverse-transcription was included as a negative control. (C) Stability of let-7d/g/i in serum after extended storage (n = 5). Serum samples were equally divided and stored at room temperature, 4°C, -20°C or -80°C for 1, 2, 3, 7, 14 or 30 days. For each time point, total RNA was isolated and let-7d/g/i was measured by RT-qPCR assay. Storage at room temperature for 30 days yielded no apparent increase in Cq values. (D) Instability of other RNAs in serum (n = 5). Serum samples were equally divided and stored at room temperature for 1 to 24 h. For each time point, total RNA was isolated, and the levels of some large molecular weight RNA (β-actin, GAPDH and 28S rRNA) and snRNA/snoRNA (U6, RNU44, RNU48, SNORD24, SNORD38B, SNORD43, SNORA66 and SNORA74A) were measured by RT-qPCR. Storage at room temperature for 24 h resulted in an apparent increase of Cq values for these RNAs. (E) Stability of let-7d/g/i in serum after RNase digestion (n = 5). Serum samples were treated with 10 U/ml RNase A and 400 U/ml RNase T1 for 4 h at 37°C. After the treatment, the RNA was extracted from the serum, and the levels of let-7d/g/i were assessed by RT-qPCR. (F) Instability of other RNAs in serum after RNase digestion (n = 5). Serum samples were treated with 10 U/ml RNase A and 400 U/ml RNase T1 for 1, 2 or 4 h at 37°C. After the treatment, the RNA was extracted and the levels of the indicated RNAs were assessed by RT-qPCR assay. (G and H) Stability of let-7d/g/i under acidic or alkaline conditions (n = 5). Serum samples were incubated for 1 h under acidic (pH 2) or alkaline (pH 12) conditions. The levels of let-7d/g/i were assessed by RT-qPCR. (I) Stability of let-7d/g/i in serum following re-freezing and re-thawing of the samples (n = 6). Serum samples were subjected to eight freeze-thaw cycles.

Mentions: We next evaluated the linear dynamic range and sensitivity of the RT-qPCR assay for measuring let-7d/g/i. Synthetic single-stranded let-7d/g/i was serially diluted and assessed by the RT-qPCR assay. Decreasing the amount of let-7d/g/i led to a corresponding increase in the mean Cq values, with a Pearson correlation coefficient (R) of 0.992 (Figure 4A). The results demonstrated that the let-7d/g/i RT-qPCR assay has a dynamic range of at least ten orders of magnitude and is capable of detecting as few as 0.01 attomole of let-7d/g/i (equivalent to 6,000 copies). Additionally, the expression levels of let-7d/g/i in RNA samples isolated from various volumes of serum were characterized using the RT-qPCR assay. The RT-qPCR assay showed excellent linearity between the serum volume and Cq value (R = 0.9865) (Figure 4B). These results demonstrate that let-7d/g/i in as little as 10 µL of serum can be efficiently detected and reliably compared across multiple samples. By referring to the standard curve, we calculated that the absolute concentration of let-7d/g/i in serum was 271.35 ± 21.48 fmol/L.


A combination of Let-7d, Let-7g and Let-7i serves as a stable reference for normalization of serum microRNAs.

Chen X, Liang H, Guan D, Wang C, Hu X, Cui L, Chen S, Zhang C, Zhang J, Zen K, Zhang CY - PLoS ONE (2013)

Characterization of the absolute concentration and the stability of let-7d/g/i in serum.(A) Dynamic range and sensitivity of the RT-qPCR assay for measuring let-7d/g/i (n = 5). Synthetic single-stranded let-7d/g/i ranging from 0.01 attomole (0.0033 attomole each, equivalent to 6×103 copies in total) to 10 pmol (3.3 pmol each) were serially diluted over ten orders of magnitude and were assessed by the RT-qPCR assay. The resulting Cq values were plotted versus the amount of input let-7d/g/i to generate a standard curve. An assay using water instead of RNA for reverse-transcription was included as a negative control. (B) Correlation of serum volume to the Cq values (n = 5). Total RNA was extracted from different volumes of serum ranging from 10 µL to 400 µL. The levels of serum let-7d/g/i were assessed by RT-qPCR. The resulting Cq values were plotted versus the serum volume used for RNA extraction. An assay using water instead of RNA for reverse-transcription was included as a negative control. (C) Stability of let-7d/g/i in serum after extended storage (n = 5). Serum samples were equally divided and stored at room temperature, 4°C, -20°C or -80°C for 1, 2, 3, 7, 14 or 30 days. For each time point, total RNA was isolated and let-7d/g/i was measured by RT-qPCR assay. Storage at room temperature for 30 days yielded no apparent increase in Cq values. (D) Instability of other RNAs in serum (n = 5). Serum samples were equally divided and stored at room temperature for 1 to 24 h. For each time point, total RNA was isolated, and the levels of some large molecular weight RNA (β-actin, GAPDH and 28S rRNA) and snRNA/snoRNA (U6, RNU44, RNU48, SNORD24, SNORD38B, SNORD43, SNORA66 and SNORA74A) were measured by RT-qPCR. Storage at room temperature for 24 h resulted in an apparent increase of Cq values for these RNAs. (E) Stability of let-7d/g/i in serum after RNase digestion (n = 5). Serum samples were treated with 10 U/ml RNase A and 400 U/ml RNase T1 for 4 h at 37°C. After the treatment, the RNA was extracted from the serum, and the levels of let-7d/g/i were assessed by RT-qPCR. (F) Instability of other RNAs in serum after RNase digestion (n = 5). Serum samples were treated with 10 U/ml RNase A and 400 U/ml RNase T1 for 1, 2 or 4 h at 37°C. After the treatment, the RNA was extracted and the levels of the indicated RNAs were assessed by RT-qPCR assay. (G and H) Stability of let-7d/g/i under acidic or alkaline conditions (n = 5). Serum samples were incubated for 1 h under acidic (pH 2) or alkaline (pH 12) conditions. The levels of let-7d/g/i were assessed by RT-qPCR. (I) Stability of let-7d/g/i in serum following re-freezing and re-thawing of the samples (n = 6). Serum samples were subjected to eight freeze-thaw cycles.
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getmorefigures.php?uid=PMC3818225&req=5

pone-0079652-g004: Characterization of the absolute concentration and the stability of let-7d/g/i in serum.(A) Dynamic range and sensitivity of the RT-qPCR assay for measuring let-7d/g/i (n = 5). Synthetic single-stranded let-7d/g/i ranging from 0.01 attomole (0.0033 attomole each, equivalent to 6×103 copies in total) to 10 pmol (3.3 pmol each) were serially diluted over ten orders of magnitude and were assessed by the RT-qPCR assay. The resulting Cq values were plotted versus the amount of input let-7d/g/i to generate a standard curve. An assay using water instead of RNA for reverse-transcription was included as a negative control. (B) Correlation of serum volume to the Cq values (n = 5). Total RNA was extracted from different volumes of serum ranging from 10 µL to 400 µL. The levels of serum let-7d/g/i were assessed by RT-qPCR. The resulting Cq values were plotted versus the serum volume used for RNA extraction. An assay using water instead of RNA for reverse-transcription was included as a negative control. (C) Stability of let-7d/g/i in serum after extended storage (n = 5). Serum samples were equally divided and stored at room temperature, 4°C, -20°C or -80°C for 1, 2, 3, 7, 14 or 30 days. For each time point, total RNA was isolated and let-7d/g/i was measured by RT-qPCR assay. Storage at room temperature for 30 days yielded no apparent increase in Cq values. (D) Instability of other RNAs in serum (n = 5). Serum samples were equally divided and stored at room temperature for 1 to 24 h. For each time point, total RNA was isolated, and the levels of some large molecular weight RNA (β-actin, GAPDH and 28S rRNA) and snRNA/snoRNA (U6, RNU44, RNU48, SNORD24, SNORD38B, SNORD43, SNORA66 and SNORA74A) were measured by RT-qPCR. Storage at room temperature for 24 h resulted in an apparent increase of Cq values for these RNAs. (E) Stability of let-7d/g/i in serum after RNase digestion (n = 5). Serum samples were treated with 10 U/ml RNase A and 400 U/ml RNase T1 for 4 h at 37°C. After the treatment, the RNA was extracted from the serum, and the levels of let-7d/g/i were assessed by RT-qPCR. (F) Instability of other RNAs in serum after RNase digestion (n = 5). Serum samples were treated with 10 U/ml RNase A and 400 U/ml RNase T1 for 1, 2 or 4 h at 37°C. After the treatment, the RNA was extracted and the levels of the indicated RNAs were assessed by RT-qPCR assay. (G and H) Stability of let-7d/g/i under acidic or alkaline conditions (n = 5). Serum samples were incubated for 1 h under acidic (pH 2) or alkaline (pH 12) conditions. The levels of let-7d/g/i were assessed by RT-qPCR. (I) Stability of let-7d/g/i in serum following re-freezing and re-thawing of the samples (n = 6). Serum samples were subjected to eight freeze-thaw cycles.
Mentions: We next evaluated the linear dynamic range and sensitivity of the RT-qPCR assay for measuring let-7d/g/i. Synthetic single-stranded let-7d/g/i was serially diluted and assessed by the RT-qPCR assay. Decreasing the amount of let-7d/g/i led to a corresponding increase in the mean Cq values, with a Pearson correlation coefficient (R) of 0.992 (Figure 4A). The results demonstrated that the let-7d/g/i RT-qPCR assay has a dynamic range of at least ten orders of magnitude and is capable of detecting as few as 0.01 attomole of let-7d/g/i (equivalent to 6,000 copies). Additionally, the expression levels of let-7d/g/i in RNA samples isolated from various volumes of serum were characterized using the RT-qPCR assay. The RT-qPCR assay showed excellent linearity between the serum volume and Cq value (R = 0.9865) (Figure 4B). These results demonstrate that let-7d/g/i in as little as 10 µL of serum can be efficiently detected and reliably compared across multiple samples. By referring to the standard curve, we calculated that the absolute concentration of let-7d/g/i in serum was 271.35 ± 21.48 fmol/L.

Bottom Line: Recent studies have indicated that circulating microRNAs (miRNAs) in serum and plasma are stable and can serve as biomarkers of many human diseases.A combination of let-7d, let-7g and let-7i is selected as a reference for the normalization of serum miRNAs and it is statistically superior to the commonly used reference genes U6, RNU44, RNU48 and miR-16.This has important implications for proper experimental design and accurate data interpretation.

View Article: PubMed Central - PubMed

Affiliation: Jiangsu Engineering Research Center for microRNA Biology and Biotechnology, State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, Jiangsu, China.

ABSTRACT
Recent studies have indicated that circulating microRNAs (miRNAs) in serum and plasma are stable and can serve as biomarkers of many human diseases. Measurement of circulating miRNAs with sufficient sensitivity and precision, however, faces some special challenges, among which proper normalization is the most critical but often an underappreciated issue. The primary aim of this study was to identify endogenous reference genes that maintain consistent levels under various conditions to serve as an internal control for quantification of serum miRNAs. We developed a strategy combining Illumina's sequencing by synthesis (SBS) technology, reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay, literature screening and statistical analysis to screen and validate the most suitable reference genes. A combination of let-7d, let-7g and let-7i is selected as a reference for the normalization of serum miRNAs and it is statistically superior to the commonly used reference genes U6, RNU44, RNU48 and miR-16. This has important implications for proper experimental design and accurate data interpretation.

Show MeSH
Related in: MedlinePlus